Shewanella phaeophyticola sp. nov. and Vibrio algarum sp. nov., isolated from marine brown algae

Abstract Two Gram-stain-negative, rod-shaped bacteria, designated as strains KJ10-1T and KJ40-1T, were isolated from marine brown algae. Both strains were catalase-positive, oxidase-positive, and facultative aerobic. Strain KJ10-1T exhibited optimal growth at 25 °C, pH 7.0, and 3 % NaCl, whereas strain KJ40-1T showed optimal growth at 25 °C, pH 7.0, and 2 % NaCl. The respiratory quinones of strain KJ10-1T were ubiquinone-8, ubiquinone-7, menaquinone-7, and methylated menaquinone-7, while the respiratory quinone of strain KJ40-1T was only ubiquinone-8. As major fatty acids, strain KJ10-1T contained C16 : 0, C17 : 1 ω8c, iso-C15 : 0, and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and strain KJ40-1T contained C16 : 0 and summed features 3 and 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The major polar lipids in strain KJ10-1T were phosphatidylethanolamine, phosphatidylglycerol, and an unidentified aminolipid, whereas those in strain KJ40-1T were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The DNA G+C contents of strains KJ10-1T and KJ40-1T were 42.1 and 40.8 mol%, respectively. Based on 16S rRNA gene sequences, strains KJ10-1T and KJ40-1T exhibited the closest relatedness to Shewanella saliphila MMS16-UL250T (98.6 %) and Vibrio rumoiensis S-1T (95.4 %), respectively. Phylogenetic analyses, based on both 16S rRNA and 92 housekeeping genes, showed that the strains formed distinct phylogenic lineages within the genera Shewanella and Vibrio. Digital DNA–DNA hybridization and orthologous average nucleotide identity values between strain KJ10-1T and other Shewanella species, as well as between strain KJ40-1T and other Vibrio species, were below the thresholds commonly accepted for prokaryotic species delineation. Based on the phenotypic, chemotaxonomic, and phylogenetic data, strains KJ10-1T and KJ40-1T represent novel species of the genera Shewanella and Vibrio, respectively, for which the names Shewanella phaeophyticola sp. nov. and Vibrio algarum sp. nov. are proposed, respectively. The type strains of S. phaeophyticola and V. algarum are KJ10-1T (=KACC 22589T=JCM 35409T) and KJ40-1T (=KACC 22588T=JCM 35410T), respectively.

In this study, two putative novel bacterial strains, designated as strains KJ10-1 T and KJ40-1 T , belonging to the genera Shewanella and Vibrio, respectively, were isolated from marine brown algae and subjected to taxonomic characterization using a polyphasic approach.
Based on this analysis, two potential novel strains, designated as KJ10-1 T and KJ40-1 T , were selected for further taxonomic characterization.Subsequently, strains KJ10-1 T and KJ40-1 T were purified by streaking on fresh MA three times.For a long-term storage, strains KJ10-1 T and KJ40-1 T were preserved at -80 °C in marine broth (MB; MBcell) supplemented with 15 % (v/v) glycerol and routinely cultured aerobically on MA or in MB for 2 days at 25 °C.To compare genomic characteristics, phenotypic properties, and fatty acid compositions, Shewanella saliphila JCM 32304 T , Shewanella algicola JCM 31091 T , Shewanella inventionis KCTC 42807 T , Vibrio hannami KACC 19277 T , Vibrio rumoiensis DSM 19141 T , and Vibrio marisflavi DSM 23086 T , obtained from their respective culture collection were used as reference strains.
The comparative analysis of 16S rRNA gene sequences revealed that strain KJ10-1 T exhibited the highest sequence similarities of 98.6, 98.0, and 97.8 % to S. saliphila MMS16-UL250 T , S. inventionis KX27 T , and S. algicola ST-6 T , respectively.On the other hand, strain KJ40-1 T showed the highest sequence similarities of 95.4, 94.8, and 94.5 % to V. rumoiensis S-1 T , V. hannami 168GH5-2-16 T , and V. marisflavi WH134 T , respectively.In the phylogenetic tree reconstructed using the ML algorithm, it was observed that strain KJ10-1 T formed a phyletic lineage with S. inventionis KX27 T with a low bootstrap value within the genus Shewanella, while strain KJ40-1 T formed a phyletic lineage with V. hannami 168GH5-2-16 T with an 84 % bootstrap value within the genus Vibrio (Fig. 1).The phylogenetic trees, reconstructed using the NJ and MP algorithms, consistently indicated that strains KJ10-1 T and KJ40-1 T formed clusters with S. inventionis KX27 T and V. hannami 168GH5-2-16 T within their respective genera, exhibiting similar tree topologies (Fig. S1, available in the online version of this article).The combined results of comparative and phylogenetic analyses based on 16S rRNA gene sequences strongly suggest that strains KJ10-1 T and KJ40-1 T are indeed members of the genera Shewanella and Vibrio, respectively.

WHOLE-GENOME SEQUENCING AND PHYLOGENETIC ANALYSIS BASED ON 92 HOUSEKEEPING GENES
The genomic DNA of strains KJ10-1 T and KJ40-1 T , as well as the reference strain V. hannami KACC 19277 T , was extracted from cells cultured in MB using the Wizard Genomic DNA purification kit from Promega, following the manufacturer's instructions.The genomic DNA was subsequently sequenced on an Oxford Nanopore MinION sequencer.The obtained sequencing reads were subjected to de novo assembly using Flye (version 2.9.1) [22], and the quality of the assembled genomes was assessed based on their completeness and contamination rates using the CheckM program (version 1.0.4)[23].
For the phylogenomic analysis of strains KJ10-1 T and KJ40-1 T , a set of 92 pre-defined single-copy housekeeping core genes was extracted from their genomes, as well as from their closely related type strains, using the bacterial core gene pipeline available at www.ezbiocloud.net/tools/ubcg[24].Subsequently, a phylogenomic ML tree with bootstrap values (1000 replications) was reconstructed based on the concatenated amino acid sequences of these core genes using the mega11 software.Additionally, the orthologous average nucleotide identity (OrthoANI) and digital DNA-DNA hybridization (dDDH) values were calculated between strain KJ10-1 T and its closely related Shewanella type strains, as well as between strain KJ40-1 T with its closely related Vibrio type strains.The OrthoANI calculations were performed using the Orthologous Average Nucleotide Identity Tool software available on the EzBioCloud server (www.ezbiocloud.net/sw/oat)[25].The dDDH calculations were performed using the Genome-to-Genome Distance Calculator version 2.1 (https://ggdc.dsmz.de/distcalc2.php)[26].
The genomes of strains KJ10-1 T , KJ40-1 T , and KACC 19277 T were sequenced with average genome coverages of 46.6×, 396.0×, and 14.0×, respectively.The de novo assembly of the genome sequencing data for strains KJ10-1 T , KJ40-1 T , and KACC 19277 T resulted in draft genomes sizes of 4.8, 4.9, and 5.4 Mb composed of three, three, and 10 contigs, respectively; the N50 values for these genomes were 4763, 3253, and 1999 kb.The 16S rRNA gene sequences in the genomes of strains KJ10-1 T , KJ40-1 T , and KACC 19277 T were identical to those obtained through PCR-based sequencing.The completeness and contamination rates of the assembled genomes for strains KJ10-1 T , KJ40-1 T , and KACC 19277 T were determined to be 96.5, 95.2, and 97.8 % for completeness, and 0.3, 4.6, and 5.2 % for contamination, respectively, which met the criteria representing generally high-quality genomes (90 % or higher completeness; 10 % or lower contamination) [27].The draft genome sequences for strains KJ10-1 T , KJ40-1 T , and KACC 19277 T have been deposited in GenBank under the accession numbers JAODOQ000000000, JAQLOI000000000, and JARQZP000000000, respectively.

GENOMIC FEATURES
The genomes of strains KJ10-1 T and KJ40-1 T , as well as the reference strains, were annotated using the NCBI Prokaryotic Genome Annotation Pipeline [28].Additionally, the carbohydrate-active enzymes (CAZymes) were analysed using the dbCAN2 meta server [29].Strain KJ10-1 T was predicted to have a total of 4464 genes, while strain KJ40-1 T had 4552 genes.Among these, 3466 and 4113 were protein-coding genes, 99 and 95 were tRNA genes, 25 and 28 were rRNA genes, and three each were non-coding RNA genes, respectively (Table 1).The DNA G+C contents, calculated based on the entirety of their respective genomes, were 42.1 mol% for strain KJ10-1 T and 40.8 mol% for strain KJ40-1 T .These values fall within the range of G+C contents observed in their closely related type strains (Table 1).A comprehensive summary and comparison of the general genomic features of strains KJ10-1 T and KJ40-1 T with those of closely related type strains from the genera Shewanella and Vibrio can be found in Table 1.
Bacteria inhabiting the phycosphere of marine algae may possess the capability to metabolize cell-wall components from the algae to support their growth.Some species of Shewanella and Vibrio have been previously reported to exhibit the ability to degrade various components of algal cell walls [30,31].Therefore, we conducted a genome-wide analysis to examine the distribution of genes encoding CAZymes, which are potentially associated with the breakdown of algal cell-wall components, in the genomes of strains KJ10-1 T and KJ40-1 T , as well as their respective reference strains.This analysis revealed a total of 68 putative CAZyme-encoding genes in the genome of strain KJ10-1 T , while strain KJ40-1 T had 88 such genes.These numbers generally exceeded those observed in the reference strains of Shewanella and Vibrio (Table 1).This suggests that strains KJ10-1 T and KJ40-1 T isolated from marine algae may possess enhanced capabilities to utilize cell-wall components from marine algae compared to other species of Shewanella and Vibrio.Notably, genes belonging to the glycoside hydrolase (GH) and glycosyltransferase (GT) families were abundantly identified among the six major CAZyme categories.Strain KJ10-1 T and its reference strains commonly had genes from the GH103, GH23, and GT51 families, which encode peptidoglycan lyase, peptidoglycan transglycosylase, and peptidoglycan glycosyltransferase, respectively.However, only strain KJ10-1 T had the polysaccharide lyase (PL) 17 family gene, which corresponds to alginate lyase, suggesting its differential ability to decompose alginate.On the other hand, strain KJ40-1 T and its reference strains commonly possess GH23 family genes corresponding to lysozyme, peptidoglycan lyase, and chitinase.However, strain KJ40-1 T did not have the GH1 family gene, corresponding to βglycoside hydrolases, unlike other Vibrio reference strains, suggesting its limited ability to decompose glycan compared to its reference strains.

MORPHOLOGY AND PHENOTYPIC PROPERTIES
The growth ability of strains KJ10-1 T and KJ40-1 T on various standard bacteriological agar media (all sourced from MBcell), including MA, Reasoner's 2A (R2A) agar, Luria-Bertani (LB) agar, tryptic soy agar (TSA), and nutrient agar (NA), was evaluated.The NaCl concentrations in R2A agar, LB agar, TSA, and NA were adjusted to 2 % (w/v).To assess growth temperature and pH, the strains were cultivated at different temperatures (4 °C, 10-40 °C at 5 °C intervals) and pH values (ranging from pH 4.0 to 11.0 in 1.0 pH intervals) on MA and in MB, respectively.For pH adjustment, sodium citrate, Na 2 HPO 4 /NaH 2 PO 4 , Tris-HCl, and NaOH buffer systems were used to prepare MB media with pH values of 4.0-5.0,6.0-7.0,8.0-9.0, and 10.0-11.0,respectively [32].The pH levels were adjusted after autoclaving (at 121 °C for 15 min).Salt tolerance was assessed in MB with varying NaCl concentrations (ranging from 0-10 % at 1.0 % intervals, w/v) prepared in the laboratory according to the MB formula: 5 g peptone, 1 g yeast extract, 0.1 g ferric citrate, 5.9 g MgCl 2 Physiological and biochemical tests for strains KJ10-1 T and KJ40-1 T were conducted using cells that were incubated at 25 °C for 2 days.Cell morphology, size, and the presence of flagella were examined through transmission electron microscopy (JEM-1010, jeol) and phase-contrast microscopy (Axio Scope.A1, Carl Zeiss).Gram staining was performed using a Gram stain kit from bioMérieux, following the manufacturer's instructions.Anaerobic growth was assessed on MA for a duration of 21 days at 25 °C under anaerobic conditions created using the GasPak Plus system (BBL).Oxidase activity was evaluated by observing the oxidation of 1 % (w/v) tetramethyl-p-phenylenediamine (Merck), while catalase activity was determined by the production of oxygen bubbles in a 3 % (v/v) aqueous hydrogen peroxide solution.The phenotypic characteristics of strains KJ10-1 T and KJ40-1 T were examined alongside reference strains under the same conditions at their respective optimal temperatures.Hydrolysis of casein (1 % skimmed milk, w/v), starch (1 %), aesculin (0.1 %), l-tyrosine (0.5 %), Tween 20 (1 %), and Tween 80 (1 %) was assessed on MA, following previously protocols [33].Additional biochemical features and enzymatic activities were evaluated using the API 20NE and API ZYM kits from bioMérieux, following the manufacturer's instructions, except that the solutions in the API kits were adjusted to approximately 2 % NaCl.
*Data marked with an asterisk were obtained from previous studies for the respective strains.
respective reference strains, shared several physiological, and biochemical features (e.g., flagellum motility, oxidase and catalase activities, nitrate reduction, and indole production for strain KJ10-1 T ; d-glucose fermentation, oxidase and catalase activities, and hydrolysis of aesculin and l-tyrosine for strain KJ40-1 T ), there were also notable differences (e.g., colony colour, growth conditions, and hydrolysis of starch, casein, l-tyrosine, and Tween 20 for strain KJ10-1 T ; colony colour, growth conditions, flagellum motility, nitrate reduction, and indole production for strain KJ40-1 T ).These differences enable the differentiation of strains KJ10-1 T and KJ40-1 T from their respective reference strains (Tables 2 and 3).
For the analysis of cellular fatty acids, the cells of strains KJ10-1 T and KJ40-1 T , along with their reference strains, were aerobically cultivated in MB at their respective optimal temperatures and harvested during their exponential growth phases (when optical density reached 0.8 at 600 nm).The cellular fatty acids were subsequently extracted from the harvested cells, saponified, and methylated following the standard midi protocol.The resulting fatty acid methyl esters were analysed using a 6890-gas chromatograph (Hewlett Packard) and identified using the RTSBA6 database of the Microbial Identification System (Sherlock version 6.0B) [35].
The polar lipids of strains KJ10-1 T and KJ40-1 T were extracted from cells harvested during their exponential growth phases and analysed according to the procedure outlined by Minnikin et al. [36], using a two-dimensional thin-layer chromatography method.The following reagents were used to identify different types of polar lipids: 10 % ethanolic molybdophosphoric acid (for total polar lipids), ninhydrin (for aminolipids), Dittmer-Lester reagent (for phospholipids), and αnaphthol/sulphuric acid (for glycolipids).The presence of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and diphosphatidylglycerol (DPG) in strains KJ10-1 T and KJ40-1 T was confirmed using standard polar lipid compounds purchased from Sigma-Aldrich.
In strain KJ10-1 T , the major cellular fatty acids (at least 10 % each) were C 16 : 0 , C 17 : 1 ω8c, iso-C 15 : 0 , and summed feature 3 (comprising C 16 : 1 ω7c and/or C 16 : 1 ω6c; Table S1); whereas in strain KJ40-1 T , the major cellular fatty acids were C 16 : 0 and summed features 3 and 8 (comprising C 18 : 1 ω7c and/or C 18 : 1 ω6c; Table S2).While the overall fatty acid profiles of strains KJ10-1 T and KJ40-1 T were generally similar to those of their closely related reference Shewanella and Vibrio strains, there were some differences in the proportions of certain fatty acids (Tables S2 and S3).For instance, C 16 : 0 and C 17 : 1 ω8c were predominantly found in strain KJ10-1 T , whereas they were detected only in trace amounts in S. saliphila JCM 32304 T .Additionally, C 12 : 0 3-OH was present in trace amounts in strain KJ10-1 T , but in other reference Shewanella strains, it was present in amounts exceeding 2 %.On the other hand, iso-C 15 : 0 , C 18 : 1 ω9c and C 18 : 1 ω7c were not detected in strain KJ40-1 T , but they were identified in V. hannami KACC 19277 T , V. rumoiensis DSM 19141 T , and V. marisflavi DSM 23086 T , although the detected amounts differed significantly among them.
The major polar lipids identified in strain KJ10-1 T were PE, PG, and an unidentified aminolipid, while those in strain KJ40-1 T were PE, PG, and DPG (Fig. S3).These results are consistent with previous reports on closely related species of Shewanella [7][8][9] and Vibrio [16][17][18], which also found PE and PG to be the main polar lipids in strain KJ10-1 T , and PE, PG, and DPG to be dominant in strain KJ40-1 T .
The type strain is KJ10-1 T (=KACC 22589 T =JCM 35409 T ), isolated from a brown alga Ishige foliacea collected in the Republic of Korea.The genome size and DNA G+C content of the type strain are 4.8 Mb and 42.1 mol% (calculated from the whole genome sequence), respectively.The GenBank accession numbers for the 16S rRNA gene and genome sequences of strain KJ10-1 T are OK626769 and JAODOQ000000000, respectively.
The type strain is KJ40-1 T (=KACC 22588 T =JCM 35410 T ), isolated from a brown alga Sargassum fusiforme collected in the Republic of Korea.The genome size and DNA G+C content of the type strain are 4.9 Mb and 40.8 mol% (calculated from the whole genome sequence), respectively.The GenBank accession numbers for the 16S rRNA gene and genome sequences of strain KJ40-1 T are OK626761 and JAQLOI000000000, respectively.

Fig. 1 .
Fig. 1.Maximum-likelihood trees based on 16S rRNA gene sequences, illustrating the phylogenetic relationships between strains KJ10-1 T (a) and KJ40-1 T (b) and their closely related taxa.The filled circles represent nodes that were also found in the neighbour-joining and maximum-parsimony trees.Bootstrap values greater than 70%, based on 1000 replicates, are indicated at the branching points.Pseudoalteromonas tetraodonis KMM 458 T (AF214729) and Salinivibrio costicola NCIMB 701 T (X95527) were used as the outgroups for the trees of strains KJ10-1 T and KJ40-1 T , respectively.The scale bars indicate 0.01 changes per nucleotide position.

Fig. 2 .
Fig. 2. Maximum-likelihood phylogenomic trees based on the concatenated amino acid sequences of 92 housekeeping core genes, illustrating the phylogenetic relationships between strains KJ10-1 T (a) and KJ40-1 T (b) and their closely related taxa.Bootstrap values greater than 70%, based on 1000 replicates, are shown at the branching points.Pseudoalteromonas tetraodonis NBRC 103034 T (BSNE00000000) and Salinivibrio costicola ATCC 33508 T (AQOF00000000) were used as outgroups for the trees of strains KJ10-1 T and KJ40-1 T , respectively.The scale bars indicate 0.05 changes per amino acid.